Secretion of heterologous proteins is a widely used technique in industry. A cell can be transformed with a nucleic acid encoding a heterologous protein of interest to be secreted and thereby produce large quantities of desired proteins. This technique can be used to produce a vast amount of protein over what would be produced naturally. Proteins of interest are proteins with a wide variety of industrial applications, including therapeutic and agricultural uses, as well as use in foods, cosmetics, cleaning compositions, animal feed, etc. Thus, increasing secretion of proteins produced by micro-organism is of general interest.
Advances in cellular and molecular biology have made it possible, in certain cases, to identify a gene encoding a desired protein, to isolate the gene, to insert the gene into a host cell and to express the inserted gene in the host cell to produce the desired protein. Bacteria have been intensively studied as host cells. When bacteria are used as host cells for this heterologous gene expression, a frequently encountered problem is however that most bacterial expression systems produce proteins intracellularly, and it is usually necessary to disrupt the cells to ensure recovery of the products.
The problem may be overcome by having the bacteria secrete the desired protein into the growth medium. One particularly well documented method of directing the secretion of proteins is the use of a secretory signal sequence. When a signal peptide is fused to the amino-terminal end of a heterologous protein, it directs the heterologous protein to the secretory machinery at the cell membrane. The heterologous protein is then translocated across the membrane. Optionally a specific protease, sometimes referred to as “signal peptidase” or “leader peptidase”, removes the signal peptide and releases the heterologous protein.
Translocation of proteins into periplasmic space or secretion into their culture media is subject to a variety of parameters. Typically, vectors for secretion of a protein of interest are engineered to position DNA encoding a secretory signal sequence 5′ to the DNA encoding the protein of interest. To increase secretion several approaches can be followed: trying several different signal sequences, mutating the signal sequence, or altering the secretory pathway within the host. However, in many cases the amount of heterologous protein secreted when making use of only a signal peptide to ensure secretion is usually very small, and a significant amount of the heterologous protein is often degraded after it is secreted.
Clostridium is a genus of Gram-positive bacteria, which is represented by a wide variety of strains. Clostridium bacteria are spore-forming anaerobic bacteria. This genus comprises solventogenic Clostridia such as C. acetobutylicum that are able to convert various sugars and polysaccharides into acids and solvents, and cellulolytic Clostridia, such as Clostridium cellulolyticum, that are able to efficiently degrade cellulose and related plant cell wall polysaccharides. More in particular, Clostridium cellulolyticum produces and secretes large cellulolytic complexes called cellulosomes that efficiently degrade cellulose and related plant cell wall polysaccharides. These complexes contain various enzymes which are tightly bound to a large protein devoid of enzymatic activity called “scaffoldin”. The binding of the enzymes on the scaffoldin occurs through interaction between cohesion modules on the scaffoldin and complementary dockerin domains on the enzymes. This high affinity interaction between dockerins and scaffoldins has been suggested for biotechnology applications e.g. recombinant protein purification (Craig et al. 2005, J. Biotechnol. 121:165-173).
On the contrary, C. acetobutylicum although it contains in its genome contains a large cluster of genes encoding cellulolytic enzymes and a scaffoldin, is not able to grow on crystalline cellulose.
One of the strategies to combine cellulose-degrading activity with solvent production in one organism has been to introduce the genes encoding the cellulosome of C. cellulolyticum into C. acetobutylicum. Mingardon et al. have demonstrated the production, assembly and secretion of a minicellulosome by Clostridium acetobutylicum by co-expressing the Mannanase gene Man5K from Clostridium cellulolyticum with the gene cipC1 encoding a truncated scaffoldin also from C. cellulolyticum therein (Mingardon et al. Applied Environm. Microbiol. 2005, vol 71(3): 1215-1222).
Several groups have investigated the possibility of increasing or improving the cellulolytic activity of cellulosome complexes by playing with the different modules present therein and combining different types of cellulases in what is referred to as “designer cellulosomes”. It was demonstrated that bifunctional and trifunctional designer cellulosomes which include a chimeric scaffoldin with two or three cohesins of divergent specificity and two or three cellulases each bearing a dockerin complementary to one of the cohesins yielded a multiprotein complex with enhanced synergistic activity on recalcitrant substrates such as straw (Fierobe et al. 2002, J. Biol. Chem. 277, 49621-19630; Fierobe et al. 2005, J. Biol. Chem. 280(16):16325-16334). In addition it was found that such cellulosomes could include combinations of bacterial and fungal enzymes (Mingardon et al. 2007, Appl. Environm. Microbiol. 73(12):3822-3832). In these experiments the cellulosomes were either produced by co-expression of the vectors encoding the different parts of the cellulosome in Clostridium cellulolyticum which naturally secretes these proteins or by mixing the recombinantly produced and purified scaffoldins and enzymes in vitro.
Mingardon et al. describes the production of a “covalent cellulosome”, which comprises, in a single polypeptide chain, a CBM together with a family 48 and a family 9 catalytic module. This protein was recovered from E. coli in which it was overexpressed by breaking the cells in a French press and purifying the recombinant protein using the c-terminal His tag. The covalent cellulosome was found to be significantly less active on Avicel substrate than the corresponding hybrid cellulosomes (Mingardon et al. 2007, Appl. Environm. Microbiol. 73(22):7138-7149).
Cloning of heterologous or homologous genes encoding secreted proteins, and (over)production and secretion of such heterologous or homologous proteins by bacterial cells such as Clostridium species other than C. cellulolyticum has not been very widely reported up until now, probably as a result of problems encountered with ensuring secretion of recombinant proteins by these hosts.
In view of the above, it is clear that there is a need in the art to improve secretion of proteins by bacterial cells.